Original Research Article

Comparative ultrastructure evaluation of particle biomaterial of ovine origin

João César Zielak1

Patrícia Locatelli1

Sônia Pozzan1

Allan Fernando Giovanini1

Tatiana Miranda Deliberador1

Cícero de Andrade Urban1

Flares Baratto-Filho1

Corresponding author:João César Zielak5,300 Professor Pedro Viriato Parigot de Souza St. – Campo CompridoZIP code 81280-330 – Curitiba – PRE-mail: jzielak2@gmail.com

1 Dental School, Positivo University – Curitiba – PR – Brazil.

Received for publication: September 13, 2010. Accepted for publication: October 7, 2010.

Abstract

Introduction: Xenogenic bone grafting biomaterials have been vastly studied. Easy clinical use and low cost have attracted professionals and patients. Alternatively to bovine biomaterial, ovine biomaterial can be developed. Objective: To compare by scanning electron microscopy the ultrastructure of a particle ovine biomaterial to equine and bovine commercially available biomaterials, besides to human bone from a tissue bank. Material and methods: Ovine mandibular bone, from a local sheep farm, was ground and submitted to a chemical and physical sterilization protocol to be compared to different biomaterial particles. Images were obtained and evaluated according to the quantity and area values of porosities (χ2), visual surface roughness, and Ca/P ratio. Results: There was no statistical difference among the sample images; however, the bovine particles presented marked porosities and roughness. Conclusions: In relation to porosity, experimental ovine bone presented closer characteristics to human bone than equine and bovine. Regarding to surface roughness, equine bone was the most similar to human bone, followed by ovine and bovine. Concerning to Ca/P ratio, ovine bone presented the lowest and most distant value from human bone, followed by equine and bovine.

Keywords: bone grafting; experimental; ultrastructure.

ISSN:Printedversion:1806-7727Electronicversion:1984-5685RSBO.2011Jan-Mar;8(1):36-42

RSBO.2011Jan-Mar;8(1):36-4237

Introduction

In Dentistry, in the event of great bone loss, grafting is essential for aesthetic and functional correction. Costa and Veinstein [4] reported the classical grafting division: 1) autogenous or self bone, i.e., when the tissue is transferred from one area (donor site) to another (receptor site) in the same subject, not provoking an immune reaction; 2) allogeneic, homogenous or homograft, i.e., tissues grafted among individuals of the same species with non-identical genes, such as fresh, frozen, lyophilized bone (FDBA), demineralized and lyophilized bone (DFDBA); 3) alloplastic, foreign body, inert, synthetically produced, used for tissue grafting, such as calcium phosphate, hydroxyapatite, bioceramic, among other types; and 4) xenografts, heterografts or heterologous, performed among individuals of different species, for example, from animal origin, such as the bovine bone, or from other animal vivarium, such as the equine bone. For each graft type, several materials are employed, generically named as biomaterial grafts.

Non autogenous materials of the aforementioned classification has evoked a great interest by the professionals, since the surgical procedure is faster, because the material to be grafted is already prepared, only requiring the receptor site. This reduces the risks due to the lack of donor site wound, being very advantageous for the patient [5, 14].

Biomaterial’s criteria selection and processing technique should be ef f icient and sa fe. By understanding the nature of the materia l’s processing, particularly the inf luence of the several techniques of biological and biomechanical preservation and/or the processes of incorporation and association among materials, each biomaterial is selected according to the surgical reconstruction goal. Currently, the availability of different biomaterials have increased, and the agility in the acquisition of treatment techniques provided an opportunity of innovative ways of surpassing the countless reconstructive challenges besides offering to many people a more satisfactorily treatment option [15].

The use of bovine bone as a source of raw material for the production of graft’s biomaterial has been studied for some time [20]. After harvesting, the bone processing is carried out in order to remove the cellular rests, lipids and proteins. It is possible that these substances, of great potential for the receptor’s immune system activation, interfered in graft’s biocompatibility. Bone treatment with peroxides for matrix decellularization helps to

maintain the collagen-apatite complex and produces a higher mechanical resistance of the obtained material [24].

Material surface is capable of influencing the cellular colonization. Marins et al. [9] characterized the particles of an organic bovine biomaterial by scanning electronic microscopy and evaluated its application in vivo (rats) by light microscopy. The results demonstrated that the studied material, with its preserved micro-architecture, is slowly resorbed and can act as a space for cellular filling, enabling the angiogenesis, cellular migration, bone adhesion and neoformation at the lesion’s edge in which it was grafted.

Similar to bovine animals, other animals reared, on a regular basis and a large scale, are a source for graft biomaterials production. By comparing, for example, the bovine to ovine cattle, some factors should be taken into consideration: both can be fed only with pasture, because they are ruminant animals; sheep adapt better to climatic conditions and require a smaller physical space of pasture; besides that, the onset of their breeding and slaughtering is between 6 and 9 months [13], while bovine, generally, need to reach 24 months [23]; also ovine’s gestation period is only five months, while bovine demands 10 months [13, 17].

The possibility of disease transmission is other relevant factor that causes apprehension when animal origin biomaterials are an issue. Among the scariest diseases regarding to either bovine or ovine animals, is the transmissible spongiform encephalopathy (TSEs), which can be caused by the accumulation of modified glycoproteins on the individual’s nervous tissues. The modified glycoproteins belong to a class of substances, so called prions. As it is known, TSEs occurring in humans have the same causative agent as bovine and ovine encephalopathies [25]; however, although prions can be resistant, there are methods to neutralize them [12].

Since study reports on ovine origin biomaterials are scarce, the aim of this study was to compare, through scanning electronic microscopy (SEM), the ultrastructure of an experimental particle biomaterial of ovine origin to commercially available equine and bovine bone and human bone particles coming from a muscle-skeletal tissue bank.

material and methodsThe ovine material was harvested from the

mandible bone of a sheep (Suffolk), 6-months old, coming from the Cabanha Kulik vivarium, located at Barreirinha neighborhood, Curitiba, Parana, Brazil.

Zielaket al.Comparativeultrastructureevaluationofparticlebiomaterialofovineorigin38–

The animal was slaughtered for feeding purposes by the vivarium’s employees, and its head was discarded. The same employee stored the sheep’s head in a freezer (-12ºC). Next, at the laboratory, the mandible was separated and had its surface cleaned by the removal of soft tissue remnants (periosteum and muscular insertion) through knife and scalpel (Duflex, Curitiba, Parana, Brazil). Following, the sheep’s mandible was washed in tap water and divided into fragments of about 2.0 x 1.0 x 0.5 cm. Some fragments were submitted to the following procedures: 1) immersion on 2% sodium hypochlorite, at 20ºC, for 12 hours, 2) washing in tap water (2 minutes), 3) immersion on 2% sodium metabisulphite solution, for 3 minutes, for hypochlorite inactivation, 4) washing in tap water (2 minutes), 5) drying through dry heat, at 20ºC, for 2 hours, 6) fragments grinding in electrical bone grinder (Kopp, Curitiba, Parana, Brazil), and 7) wet heat (autoclave), for 20 minutes, at 135ºC. By using the aforementioned procedures, a small bone fragment was grinded, coming from a surgical procedure and stored in the Muscle-skeletal tissue Bank of the Clinic Hospital of the Federal University of Parana.

Next, some ovine and human bone particles were stored in a desiccator with silica, mounted on a SEM support, and metalized (Baltec, Balzers, Germany). Other fragments of the commercially available biomaterials were also metalized and mounted on the support: equine (Spongy BIO-GEN BGS-05, Bioteck, Chieri, Torento, Italy) and bovine bone (Genox Inorg Spongy, Baumer, Mogi-Mirim, Sao Paulo, Brazil). The supports were taken into electronic microscopy for chemical and ultrastructural morphological evaluation of the surface (electrons dispersive energy spectroscopy) (SEM-EDS, JSM, 6360-LV, Jeol, Japan). The obtained images were analysed and compared by the aid of image analysis software (Image Tool 3.00, University of Texas Health Science Center, USA).

Results

Through SEM image analysis, the visual aspect of the superficial roughness and porosity of the particles was observed at x 30, 150, 1,500, and 10,000 magnification (figure 1).

Bovine bone®

figure 1–Imagesofthestudiedbiomaterials’surface

Human bone Ovine bone

Equine bone®

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Median25%-75%Min-Max

Por

osit

y ar

ea

human bone ovine bone equine bone® bovine bone®

The pores amount and their areas were measured. Data statistical analysis (χ2) demonstrated significant differences among the experimental and the commercially available bone grafts (equine and bovine); however, there was no expressive difference in relation to human bone (table I and graph 1).

Table I–Comparativeanalysis(χ2)oftheamountandareaoftheporesonthebiomaterialsurfaces

Human bone Bovine bone Equine bone® Bovine bone®

Human bone 1.000000 0.040612 0.001803

Ovine bone 1.000000 0.085828 0.007441

Equine bone 0.040612 0.085828 1.000000

Bovine bone 0.001803 0.007441 1.000000 0.001803

Graph 1 –Comparativeoftheporosityareas(µm2)amongthebiomaterials

Table II shows the Ca/P relationship in the studied biomaterials, which evidenced the following values: 3.3 for human bone, 2.0 for ovine bone, 2.7 for equine bone, and 2.9 for the bovine specimen.

Table II –Distribution,inweight(%),ofcalcium(Ca),phosphorus(P)ontheparticlesurfacesofbiomaterialsbyEDSanalysis(dispersiveenergyspectroscopy)

Human bone Ovine bone Equine bone® Bovine bone®

Ca 28.17 49.32 37.12 34.19

P 8.41 23.82 13.66 11.50

DiscussionThe choice for a raw material source to produce

a determined product should take into consideration several factors, among them, costs, productivity, and mainly, sustainability. Considering the animal models of extensive livestock already inserted into our society, ovine seem to be ahead of bovine cattle, since in a same space amount, they can produce more animals, in less time period. Consequently, we can hypothesize that less deforestation may occur, which has been one of the biggest causes of environmental devastation, nowadays [16, 21].

Although this study’s goal was not to characterize the prions presence in the evaluated particles, the presented protocol considered the current regulation, aiming to biosecurity in the xenogenic biomaterials production: use of bone as raw material, a prion low-infection tissue; and processing capable of prion inactivation [3]. Therefore, we proposed an association of chemical and physical techniques for prion inactivation, according to the guidelines indicated for international organizations [12], by employing disinfection by sodium hypochlorite oxidation and wet heat application (autoclave).

Zielaket al.Comparativeultrastructureevaluationofparticlebiomaterialofovineorigin40–

Ogurtan et al. [11] reported the particle bovine bone superiority used in experimental defects in vivo. In this investigation, particle size was not evaluated after the electrical grinding. In clinical practice, other types of grinders are available: manual pestle, and ratchet. An interesting feature of the electric grinder is to produce chip particles, with small thickness (0.5-1 mm, according to the manufacturer), which occupy a relatively large area. Despite of the area ranging, the electric grinder produces fragments with more regular aspect than particles obtained by either the ratchet or manual pestle grinders, frequently used in surgical techniques [10]. Concerning to the particles of this study’s commercially available materials, according to their manufacturers, both have 0.5 and 1-mm size.

Generally, the commercially bovine bone’s ultra-structure demonstrated the most different features in relation to other materials: large porosity and structure’s aspect similar to several aggregates of small unities, probably hydroxyapatite, i.e., the bovine bone’s inorganic part that remained after the bioprocessing action to which the specimen was submitted (figure 1).

On the other hand, regarding to the visual aspect of the surface roughness (figure 1), the equine specimen was the most similar to human bone. According to its manufacturer, this bone was subjected to an enzymatic processing, at 37ºC, favoring the maintenance of a surface more similar to the in vivo situation. However, Śmieszek-Wilczewska et al. [22] assured that the equine bone bioprocessing reaches a temperature of 125ºC. In general, still in relation to the superficial roughness visually observed and considering a less similarity to human bone, equine bone was followed by the experimental ovine specimen and bovine bone.

The quantitative analysis revealed porosities with irregular sizes and amounts (table 1 and graph 1), demonstrating a greater range in the bovine bone particles. The presence of pores in the materials may be important, because cellular colonization of bone tissue is facilitated by their presence. The pores should present sizes varying from 50 to 100 micrometers in order to result in good vascularization and cellular colonization, for reducing the infection risks [26]. Leonel et al. [7] described the importance of the porosities on biomaterial surfaces, due to be an elemental condition for their stabilization and incorporation by live tissues. The porosity existence on graft materials, its diameter, morphology, and the presence of intercommunications are essential features that help cellular migration within the

graft, allowing (or not) bone neoformation. This study’s bovine bone presented porosities with areas very superior to 100 micrometers square, while the other biomaterials (equine, experimental ovine, and human bone) demonstrated lesser porosities with similar sizes (graph 1).

Since the porosity sizes may influence on graft vascularization, it can be understood that the greater the biomaterial’s porosity, the greater the possibility of absorption of biomaterial particles, and consequently, a greater substitution by newly-formed bone, which was the case of this study’s bovine bone. Other study on bovine bone showed that the deproteinization used in the processing (up to 1,000ºC) is also capable of modifying the biological response and that this processing would favor bone repair [14]. On the other hand, a great increase of temperature generates permanent modifications on bone matrix’s structure and crystallinity [8], which can reduce its absorption at the graft site and help bone substitution. Besides that, it potentially contributes to the bone neoformation with less mechanical resistance due to the presence of the inert particles involved by conjunctive tissue and which did not participate in tissue remodeling, a very important particularity concerning to bone renovation [19].

Concerning to biomaterial’s Ca/P relationship, it is known that it can influence on repair quality. Such relationship is directly linked to the solubility in physiologic medium, and the lesser the Ca/P ratio, the greater the biodegradation. For example, biomaterial particles such as calcium triphosphate show a Ca/P ratio of 1.50, while pure hydroxyapatite Ca/P ratio is 1.67 [27]. Even with the greatest Ca/P amounts (table II), the experimental ovine bone showed the lowest Ca/P ratio. This can result in a greater biodegradation degree than the other biomaterials of the sample. Even so, further investigations should be carried out in order to observe the legitimacy of the bioprocessing here exposed. The study of the micro-architecture of a determined biomaterial needs to be functionally evaluated, aiming to know other features relevant to cellular colonization. In vitro researches, for example, which prove both the efficacy in prions inactivating and the cellular culture in the presence of the ovine biomaterial would be future perspectives. It is already known that the free oxygen generated by the peroxides decomposition [18], the temperature and pressure variations [1] and the radiation [2] of the physical and chemical processes [6] of sterilization morphologically alter several biomolecules, such as proteins, lipids, amino acids, carbohydrates, and nucleic acids, which through feedback process,

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influence the behavior of the cells and the tissues exposed to biomaterials. Because of that, in vivo experimental trials are also important, providing relevant data on the biocompatibility and bone tissue reaction for clinical application of the experimental ovine biomaterial.

Conclusion

According to this study’s limitations, it can be concluded that:• concerning to porosity (amount and area), the

experimental ovine bone showed characteristics more similar to human bone than equine and bovine bone;

• concerning to visual roughness, equine bone was closer to human bone, followed by ovine and bovine bone;

• concerning to Ca/P ratio, ovine bone evidenced the lowest and farthest value from human bone, followed by equine and bovine bone.

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How to cite this article:

Zielak JC, Locatelli P, Pozzan S, Giovanini AF, Deliberador TM, Urban CA et al. Comparative ultrastructure evaluation of particle biomaterial of ovine origin. RSBO. 2011 Jan-Mar;8(1):36-42.